In previous studies, we introduced a doxycycline-inducible PAX3-FOXO1 expression construct into immortalized human myoblasts (with and without a constitutive MYCN expression construct) to generate a cell culture system in which PAX3-FOXO1 expression can be up-regulated by doxycycline treatment and then down-regulated by doxycycline withdrawal. In cell culture studies, oncogenic transformation was detected in cells with PAX3-FOXO1 and MYCN expression, but not in cells only expressing PAX3-FOXO1 alone. In animals receiving an intramuscular injection, progressively growing tumors formed from PAX3-FOXO1-inducible myoblasts when treated with doxycycline; cells expressing both PAX3-FOXO1 and MYCN formed tumors several weeks earlier than cells expressing PAX3-FOXO1 without exogenous MYCN. When doxycycline was withdrawn, tumor formation stopped and regressed, and then progressively growing tumors re-formed several weeks later in the continued absence of doxycycline. During the past year, we analyzed the histopathology and expression of specific proteins in the primary and recurrent tumors formed in the animal studies. All tumors showed histologic resemblance to human ARMS with high expression of MyoD and myogenin. Frequent mitoses and a high Ki67 staining index were indicative of a high proliferative rate. Western blot analysis of the regressing tumors showed a rapid decrease in PAX3-FOXO1 protein expression. Microscopic examination of regressing lesions showed a striking increase in multinucleated giant cells, indicative of myogenic differentiation. In many giant cells, the presence of pyknotic nuclei and a large transient increase in caspase-3 staining provided evidence of superimposed cell death. There was also a transient decrease in Ki67 staining indicative of a block in proliferation. Western blot analysis of recurrent tumors showed that half of these tumors have no detectable PAX3-FOXO1 protein expression. One recurrent tumor contained a large dominant nodule of densely packed undifferentiated small cells surrounded by loosely packed differentiated myotubes. This nodule had a high Ki67 staining index consistent with proliferative activity. In other recurrent tumors, there was a patchy distribution of regions with small undifferentiated cells and high Ki67 staining. These findings emphasize the in vivo role of PAX3-FOXO1 in stimulating growth and inhibiting differentiation and cell death, and suggest there may multiple mechanisms underlying formation of recurrent tumors. In cell lines derived from primary and recurrent tumors, doxycycline addition induced PAX3-FOXO1 expression and withdrawal resulted in loss of PAX3-FOXO1 expression. These findings indicate that the inducible system is intact during tumorigenesis and recurrence. In focus formation assays, doxycycline induced significant focus formation in cell lines from primary and recurrent tumors. In the absence of doxycycline, one of the recurrent tumor-derived lines showed PAX3-FOXO1-independent focus formation. For the cells transduced with PAX3-FOXO1 but not MYCN, the tumor-derived cells were transformed whereas the parental cells were not transformed, suggesting selection of cells with a change that substitutes for MYCN during tumorigenesis.